Pigment

ABSTRACT

This invention relates to a new pigment in feed for salmonids, a new feed comprising this pigment and use of this pigment. The pigment comprises a diester of predominantly (3R,3′R)-astaxanthin, cantaxanthin or other carotenoids that can be used for pigmentation of salmonids prepared with an omega-3 fatty acid and/or a short chain carboxylic acid. By this invention a pigment for feed to salmonids that is more or as stable as, and biologically more effective than free astaxanthin and previously known diesters of astaxanthin and commercially available astaxanthin and cantaxanthin products, is provided. The said diesters are also useful for enhancing the growth of farmed fish, as a growth-enhancing agent in feed for farmed fish, as an appetizer in feed for fish as well as for increasing the utilization of the feed for farmed fish, and for optimising health and well-being of farmed fish.

This invention relates to a new pigment in feed for salmonids, a newfeed comprising this pigment and use of the pigment. This pigment isalso useful as an agent for enhancing the growth of farmed fish.

In feed for farmed salmonids pigment has to be added to obtain thedesired colour of the fish flesh. The pigment most commonly used isastaxanthin, but other pigments like for instance cantaxanthin, may beemployed. These pigments are all carotenoids. Such pigments are veryunstable with regard to exposure to air and elevated temperatures. Thepigments are therefore to a certain extent degraded during feedprocessing and storage.

Commercially available astaxanthin products are furthermore veryexpensive and their biological retention is very low. Astaxanthin is asmentioned above a rather unstable compound, which of course is a furtherdrawback. The low stability of astaxanthin is due to oxidation.Commercial pigment products are formulated in order to avoid or reduceoxidation. One typical formulation for astaxanthin is with gelatine andstarch. The formulations used are often, however, not optimal withrespect to biological availability of the pigment.

In Norwegian Patent No. 309386 (NO-309386) a new pigment that to someextent solved the above given problems was disclosed. This pigmentcomprises a diester of astaxanthin prepared with a carboxylic acid,wherein the carboxylic acid is an omega-3 fatty acid and/or a carboxylicacid having from 1-12 carbon atoms. A feed for salmonids comprising thesaid diester of astaxanthin, and the use of the said diester ofastaxanthin as a pigment in feed for salmonids are also disclosed inNO-309386.

In Norwegian Patent Application No. 20013354 (NO-20013354) the use ofthe diester of astaxanthin from NO-309386 for enhancing the growth offarmed fish is disclosed.

It is expected that diesters of cantaxanthin and other carotenoidsprepared with the same carboxylic acids as defined in NO-309386 andNO-20013354 will give similar effects as described in the two saidpatent specifications when used as pigments and growth enhancers,respectively.

By NO-309386 a more stable and more biologically available pigment thanfree astaxanthin and other commercial pigment products was found. Eventhough the pigment according to NO-309386 is an improvement compared tofree astaxanthin and other commercial pigment products, it is notoptimal, and it is still a strong desire and need in the aquacultureindustry to find stable and even more biologically effective pigmentsuseful in production of feed for salmonids.

Astaxanthin has two asymmetric carbon atoms at the 3 and 3′ positionsand can exist as three optical isomers; the enantiomers (3R,3′R) and(3S,3′S), and the meso form (3R,3′S) (FIG. 1).

Chemical synthesis gives equal mixtures of these optical isomers.Commercially manufactured synthetic astaxanthin, which currently is theform of the pigment predominantly added to the feed in salmonidaquaculture, thus is a mixture of the (3R,3′R)-, meso-, and(3S,3′S)-astaxanthin in the approximate ratio of 1:2:1. Astaxanthin fromnatural sources, on the other hand, varies widely in the composition ofoptical isomers, depending on the source in question. The predominantisomer in the algae Haenzatococcus pluvialis is (3S,3′S), while theyeast Phaffia rhodozyma mainly has (3R,3′R)-astaxanthin (Johnson, E. A.and An, G. H., CRC Critical Reviews in Biotechnology 11 (1991) 297).

It is generally agreed that when fed a diet containing astaxanthin inthe free form, i.e. unesterified, the optical isomers of astaxanthin areequally well absorbed and deposited in the flesh of salmonid fishes(Foss, P. et al., Aquaculture 41 (1984) 213-226; Kamata et al., NipponSuisan Gakkaishi 56 (1990) 789). The salmonids may, on the other hand,is display a certain selectivity with regard to absorption anddeposition of astaxanthin optical isomers when fed a diet containing theester form of the pigment. It is known that salmonids utilise a diester,dipalmitate, of (3R,3′R)-astaxanthin better than dipalmitate of(3S,3′S)-astaxanthin (Torrissen, O. J. et al., CRC Critical Reviews inAquatic Sciences 1 (1989) 209; Foss, P., et al., Aquaculture 65 (1987)293; Katsuyama et al., Comp. Biochem. Physiol. 86B (1987) 1; Schiedt,K., et al., Pure & Appl. Chem. 57 (1985) 685). However, wild salmon hasapproximately the same astaxanthin stereoisomer distribution in theflesh as what is present in their food, even though the food mainlycontains diesters (Lura, H., et al., Can. J. Fish Aquat. Sci., 48 (1991)429; Turujman, S. A., et al., JAOAC 80 (1997) 622). This suggests thatthe potential isomeric effect is moderate.

As described in Example 1 below, hydrolysis experiments were performedwith a diester of synthetic astaxanthin with omega-3 fatty acids and acrude enzyme preparation from salmon intestines in the same way asdescribed before (NO-309386). The reaction products were analysed withregard to stereoisomer composition. The results are given in Table 1.

EXAMPLE 1

A diester of astaxanthin was prepared by conventional chemical synthesisfrom commercially obtained synthetic astaxanthin and an omega-3 fattyacid concentrate containing more than 90% omega-3 fatty acids, mainlyEPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). Theastaxanthin used had been prepared by chemical synthesis, and thedistribution of the optical isomers (3R,3′R), meso and (3S,3′S) was25.9:50.2:23.9, respectively, determined as described for the diolfraction.

The starting astaxanthin diester was treated with a freshly preparedenzyme mixture from the intestines of recently fed salmon (Salmo salar)for 48 hours. The pigment was then separated into three fractions bypreparative thin-layer chromatography; a diol fraction with bothhydroxyl groups hydrolysed, a monoester fraction with one hydroxyl grouphydrolysed, and a remaining diester fraction. The astaxanthin in thediol fraction was converted into the corresponding diesters of(−)-camphanic acid by reaction with (−)-camphanoyl chloride, and thedistribution of astaxanthin optical isomers were determined byhigh-performance liquid chromatography (HPLC) of the dicamphanates. Theastaxanthin in the monoester fraction was first converted to diol in ahydrolysis reaction catalysed by the enzyme cholesterol esterase andsubsequently treated with (−)-camphanic acid and analysed as describedabove. Attempts to convert the astaxanthin in the remaining diesterfraction to diol using cholesterol esterase were not successful, ascomplete conversion was not obtained. The distribution of opticalisomers of astaxanthin in the remaining diester fraction was thereforenot determined.

The salmon intestine enzyme mixture displayed an unexpectedly highenantioselectivity toward the R-configuration of the astaxanthin (Table1). The astaxanthin diol fraction, i.e. free astaxanthin, had almostexclusively the (3R,3′R)-configuration, with traces of the meso-form.The monoester fraction contained predominantly the meso-form ofastaxanthin. The distribution of astaxanthin optical isomers in theremaining unhydrolysed diester fraction was not obtained, butconsidering the composition of the starting diesters, the diol and themonoester fractions, it is highly likely that the remaining diesterfraction predominantly has the (3S,3′S)-form of astaxanthin. Therelative molar amounts of pigment in the different fractions were inaccordance with what was expected based on the distribution of opticalisomers. TABLE 1 Results from the study of the enantioselectivity ofenzymes from salmon intestines for the hydrolysis of astaxanthin omega-3fatty acid diester. Pigment fraction (3R,3′R) meso (3S,3′S) Startingdiester 25.9 50.2 23.9 Monoesters (after enzyme treatment) 3.5 93.1 3.4Diol (after enzyme treatment) 94.6 5.4 ND^(a)^(a)ND; not detected

Based on the literature cited above, one could expect some degree ofenantioselectivity towards the R-configuration. However, as the isomercomposition of astaxanthin in wild salmon is approximately the same asin the food, the extreme specificity that has been demonstrated with theesters of the present invention is highly surprising.

This unexpected finding is very important. From NO-309386 it is knownthat there is a relationship between increased enzymatic hydrolysis andincreased deposition of astaxanthin in salmon muscle. The very differentrate of hydrolysis between the (3R,3′R)- and (3S,3′S)-isomers that isdemonstrated in the present invention, shows that astaxanthin diesterbased on the (3R,3′R)-isomer will have a significantly higher biologicaluptake than a diester based on the (3S,3′S)-isomer. As the personskilled in the art will know, a high biological uptake of astaxanthinindicates good pigmentation effect.

An astaxanthin diester product based on a purified stereoisomericcomposition will obviously be more expensive than a racemic or lesspurified product. The indications in the literature regarding a certainpreference of uptake of the (3R,3′R)-isomer have not been so as tosuggest to those skilled in the art that the high cost of producing a(3R,3′R)-diester would be compensated for by increased bioavailability.This is underlined by a statement from a producer of astaxanthin fromthe yeast Phaffia rhodozyma. Astaxanthin from P. rhodozyma is known tocontain mainly the (3R,3′R)-isomer. The producer states that a benefitfrom using the Phaffia product is that it contains mainly unesterifiedastaxanthin, which is known to be utilised better than esterifiedastaxanthin (Igene Biotechnology Inc.'s brochure: ‘AstaXin® Naturally!’,which was distributed at the Aquanor exhibition in Trondheim August2001). On the contrary, the present inventors have shown that theproduction of a diester of (3R,3′R)-astaxanthin containing omega-3 fattyacids, will give a significantly higher uptake than what is found withthe (3S,3′S) esterified product. The same effect will of course beobserved by using a diester of synthetic or natural(3R,3′R)-astaxanthin. Based on the teaching of NO-309386, the inventorshave assumed that utilisation of a diester of (3R,3′R)-astaxanthin and ashort chain fatty acid will have similar benefits.

Astaxanthin is very expensive, and the addition of astaxanthin or othercarotenoids is assumed to be the highest cost factor in the productionof salmon feed. The present invention shows that it will be ofcommercial value to produce a pigment that consist of the diester of(3R,3′R)-astaxanthin with a carboxylic acid, wherein the carboxylic acidis an omega-3 fatty acid and/or a short chain acid.

For simplicity, in the following the wording “omega-3 fatty acid” isalso used to denote a concentrate of omega-3 fatty acids. This will beobvious for the person skilled in the art.

It is a main object of the invention to provide a pigment for feed tosalmonids that is stable and more biologically effective than previouslyknown pigments for salmonids.

Another object of this invention is to provide a pigment that can beadded to the feed in less amounts than previously known pigments andstill give a satisfactory pigmentation of the flesh. This and otherobjects are achieved by the attached claims.

A preferred embodiment of the present invention is a diester of(3R,3′R)-astaxanthin wherein the diester is prepared with an omega-3fatty acid comprising a total amount of eicosapentaenoic acid (EPA)(all-cis C20:5 n-3) and/or docosahexaenoic acid (DHA) (all-cis C22:6n-3) from 18 to 100%.

A more preferred embodiment of the present invention is a diester of(3R,3′R)-astaxanthin wherein the diester is prepared with an omega-3fatty acid comprising a total amount of eicosapentaenoic acid (EPA)(all-cis C20:5 n-3) and/or docosahexaenoic acid (DHA) (all-cis C22:6n-3) from 40 to 100%.

Another preferred embodiment of the present invention is a diester of(3R,3′R)-astaxanthin wherein the diester is prepared with an omega-3fatty acid comprising an amount of eicosapentaenoic acid (EPA) (all-cisC20:5 n-3) from 8 to 98% and/or an amount of docosahexaenoic acid (DHA)(all-cis C22:6 n-3) from 8 to 98%.

A more preferred embodiment of the present invention is a diester of(3R,3′R)-astaxanthin wherein the diester is prepared with an omega-3fatty acid comprising an amount of eicosapentaenoic acid (EPA) (all-cisC20:5 n-3) from 25 to 98% and/or an amount of docosahexaenoic acid (DHA)(all-cis C22:6 n-3) from 15 to 98%.

Still another preferred embodiment of the present invention is a diesterof (3R,3′R)-astaxanthin wherein the diester is prepared with an omega-3fatty acid comprising approximately 50% eicosapentaenoic acid (EPA)(all-cis C20:5 n-3) and approximately 35% docosahexaenoic acid (DHA)(all-cis C22:6 n-3).

Still another preferred embodiment of the present invention is a diesterof (3R,3′R)-astaxanthin wherein the diester is prepared with a shortchain carboxylic acid being formic acid.

The astaxanthin product according to the present invention may beproduced from free astaxanthin that is obtained by chemical, biochemicalor enzymatic syntheses. Preferably, the astaxanthin used for producingthe astaxanthin products according to the present invention is obtainedfrom natural sources.

The fungus Phaffia rhodozyma is known to produce high degree of(3R,3′R)-astaxanthin in non-esterified form. A preferred embodiment ofthe present invention is therefore a diester of (3R,3′R)-astaxanthin asdefined above prepared from astaxanthin produced by P. rhodozyma.

The astaxanthin product according to the present invention comprises adiester of predominantly (3R,3′R)-astaxanthin prepared with a carboxylicacid wherein the said carboxylic acid is an omega-3 fatty acid and/or acarboxylic acid having from 1-12 carbon atoms.

Preferably the astaxanthin product comprises a diester of 50-100%(3R,3′R)-astaxanthin, more preferred the astaxanthin product comprises adiester of 80-100% (3R,3′R)-astaxanthin, and most preferred theastaxanthin product comprises a diester of 90-100% (3R,3′R)-astaxanthin.

1. A pigment for fish feed comprising a diester of predominantly(3R,3′R)-astaxanthin, cantaxanthin or another carotenoid that can beused for pigmentation of salmonids, wherein the diester is prepared withone or more carboxylic acids, selected from the group consisting ofomega-3 fatty acid and carboxylic acid having 1-12 carbon atoms.
 2. Thepigment according to claim 1, wherein the carotenoid component of thediester is 50-100% (3R,3′R)-astaxanthin.
 3. The pigment according toclaim 1, wherein the carotenoid component of the diester is 80-100%(3R,3′R)-astaxanthin.
 4. The pigment according to claim 1, wherein thecarotenoid component of the diester is 90-100% (3R,3′R)-astaxanthin. 5.The pigment according to claim 1, wherein 18 to 100% of the omega-3fatty acid is either eicosapentaenoic acid (EPA) (all-cis C20:5 n-3) ordocosahexaenoic acid (DHA) (all-cis C22:6 n-3) or a mixture of both. 6.The pigment according to claim 1, wherein 40 to 100% of the omega-3fatty acid is either eicosapentaenoic acid (EPA) (all-cis C20:5 n-3) ordocosahexaenoic acid (DHA) (all-cis C22:6 n-3) or a mixture of both. 7.The pigment according to claim 1, wherein 8 to 98% of the omega-3 fattyacid is either eicosapentaenoic acid (EPA) (all-cis C20:5 n-3) ordocosahexaenoic acid (DHA) (all-cis C22:6 n-3) or a mixture of both. 8.The pigment according to claim 1, wherein either (a) from 25 to 98% ofthe omega-3 fatty acid is eicosapentaenoic acid (EPA) (all cis C20:5n-3), (b) from 15 to 98% of the omega-3 fatty acid is docosahexaenoicacid (DHA) (all cis C22:6 n-3) from 15 to 98%, or (c) at least 25% ofthe omega-3 fatty acid is eicosapentaenoic acid (EPA) (all-cis C20:5n-3) and at least 15% of the omega-3 fatty acid is docosahexaenoic acid(DHA) (all-cis C22:6n-3).
 9. The pigment according to claim 1, whereinthe omega-3 fatty acid comprises approximately 50% eicosapentaenoic acid(EPA) (all-cis C20:5 n-3) and approximately 35% docosahexaenoic acid(DHA) (all-cis C22:6 n-3).
 10. The pigment according to claim 1, whereinthe carboxylic acid having from 1-12 carbon atoms is formic acid. 11.The pigment according to claim 1, wherein the diester is prepared fromastaxanthin obtained from a natural source.
 12. The pigment according toclaim 11, wherein the natural source is Phaffia rhodozyma.
 13. A feedfor salmonids comprising 25-70% by weight of proteins, 5-60% by weightof lipids, 0-40% by weight of carbohydrates, and pigment, in combinationwith 0-15% by weight of one or more additional components selected fromthe group consisting of fillers, adhesives, preservatives, vitamins andminerals, wherein the pigment comprises a diester of predominantly(3R,3′R)-astaxanthin, cantaxanthin or another carotenoids carotenoidthat can be used for pigmentation of salmonids, wherein the diester isprepared with one or more carboxylic acids selected from the groupconsisting of omega-3 fatty acids short chain carboxylic acids.
 14. Aprocess of raising farmed fish comprising feeding to the fish a feedthat contains a diester of predominantly (3R,3′R)-astaxanthin,cantaxanthin or another carotenoid that can be used for pigmentation ofsalmonids, wherein the diester is prepared with one or more carboxylicacids selected from the group consisting of omega-3 fatty acids andshort chain carboxylic acids. 15-21. (Cancelled)
 22. The process ofclaim 14, wherein the fish are salmonids.